SMALL CELL LUNG CANCER

Molecular Pathology OF THE

SMALL CELL LUNG CANCER

INTRODUCTION

The small cell lung cancer is an epithelial malignant tumor made up of small cells, with cytoplasm poor, marginal, with fine, granular appearance and lack of nucleoli. It has a high rate of mitosis and extensive necrosis. It differs through a very high nuclear-cytoplasmic ratio (1).

So far no precursors are not known, as well as for the lung cancer with neuroendocrine cells. The diffuse neuroendocrine hyperplasia is not considered pre-neoplastic lesion (1).

The lung neuroendocrine neoplasms are a wide range of entities phenotypic, biologically distinct, starting from the typical and atypical carcinoids up to small cell lung cancer and lung cancer with neuroendocrine cells.

The neuroendocrine appearance is highlighted through immuno-histochemistry and electron microscopy (2).

Small cell lung cancer forms simultaneously part of the neuroendocrine tumors with neuroendocrine cell
lung cancer and typical and atypical carcinoids (3.4). Small cell lung cancer morphologically differs from neuroendocrine cell lung cancer but chemotherapy for small cell lung cancer has proven effective for lung cancer with neuroendocrine cells. They are very strong entities.

Small cell lung cancer is a disease which responds well to chemotherapy, being able to reach a favorable response rate of 70% -80% of cases, of which over a third are complete responses (5). But instead the survival duration is short and 75% -80% succumbs it in the first 2 years.

But from the 2004 WHO classification of tumors resulted the oat-cell variant due to the lack of clinical and therapeutic differences (3.4). Sometimes the lesions are mixed and 28% of the surgical specimens have also shown a cancer component of non-small cell. It is necessary that at least 10% of lesions be of pure type small cell cancers in order to name it small cell cancer (5).

Regarding the susceptibility of small cell cancer occurrence, it was noticed that the genetic variations of mi-RNA (micro-RNA), which performs the control on the target genes, produce alterations of the individual susceptibility in small cell lung cancer.

Thus have been identified 2SNP (single-nucleotide polimorphism): rs3134615 and rs2291854 located in an area of the MYCL1 gene and hASH-1 neural complex of development (human Achaete-scute homlog-1). It has been identified that the alleles of the rs3134615 of the SNP is associated with the increased risk for small cell cancer, probably by attenuation of interaction with miRNA with hsa-miR-1827 leads to an altered gene expression of MYCL1 (6)

DATA ON CELL DIFFERENTIATION

WITH  DIAGNOSTIC VALUE

The small cell lung cancer, by definition, is a proliferation that arises from the bronchial basal cells and expresses low molecular weight cytokeratin.

The micro-cell carcinomas have a high mitotic rate of the proliferation and can be distinguished by determining the Ki-67 index (proliferation index). The small cell carcinoma is in fact an aneuploid neoplasm.

Another molecular signature TTF-1 (thyroid transcription factor-1). It is present in high neuro-endocrine carcinomas and not in carcinoid tumors (7).

The presence of TTF-1 helps in the differential diagnosis with basaloid carcinoma (subset of the large cell lung cancer), which is not the expression of TTF-1 (8.9).

The differential diagnosis is necessary to be established between small cell cancer and lung or chest neuroectodermal tumors. Differentiation is performed by the fact that these latter tumors have a specific marker CD99 (MIC2) and do not express cyto-keratin and nor TTF-1 (8.9). However 25% of cases of small cell cancer show the marker CD99 (10).

Small cell cancer also shows 3 neuroendocrine markers: chromogranin synaptophysin şi CD56 (neural  cell adhesion moleculeNCAM). CD56 is  considered to be the most sensitive marker in small cell cancer (11).

The disease severity and aggressiveness particularly requires an early diagnosis. Recently (2011) has been studied the presence of autoantibodies compared to 6 tumor-related antigens:p53,CAGE,NY-Eso-1, GBU 4-5,ANNEXIN-1, SOX-2 and HU-D. In 55% of cases have been found - at least in an antigen of the 6 tumor antigen - autoantibodies. In 4 of the patients have been found autoantibodies three months before being discovered the tumor. There have not been found autoantibodies compared to ANNEXIN in any case of small cell cancer, which may have a diagnostic value. The autoantibodies frequency varied between 4% for GBU 4-5 and 35% for SOX2.

The test is required for detecting small cell cancer in patients with high risk (12).

MOLECULAR genetic changes involved in the pathogenesis of small cell cancer

THE SHH (SONIC HEDGEHOG) PATHWAY

This signaling pathway induces the neuroendocrine differentiation through the making and reception of SHH signals in the epithelial stem cell compartment. The cancer cell lines with small cell express SHH. SHH fixing on the PATCH receiver leads to the activation of GLI 1 transcription factor.

This signaling pathway is activated in small cell cancer and is involved in the malignant phenotype. The SHH pathway can be blocked by means of a chemical substance, CYCLOPAMINE (extract of VERATRUM), which blocks the cell proliferations (13).

THE hASH-1EXPRESSION OF THE TRANSCRIPTION FACTOR

(HUMAN ACHAETE-SCUT HOMOLOGUE-1)

hASH-1 is a transcription factor and plays an essential role in the neural-endocrine determination and differentiation in the normal development of the nervous system, of the endocrine endoderm cells (14,15) (16,17).

In small cell cancer, non-small cell cancer with neuroendocrine component and in bronchial carcinoid tumors, hASH -1 is found at a high and uniform level (18). hASH-1 promotes the epithelial airway proliferation and potentiates tumorigenesis in the event of losing the p53 and pRb functions (16).

Data suggest that hASH -1 expression in tumors with neuroendocrine component imitate (repeat) the development manner during embryonal period (1). It intervenes in determining the tumor phenotype, but does not interfere with its maintenance (1).

THE p53 PATHWAY ALTERATIONS

The p53 gene mutations are a frequent abnormality in human cancers, including lung cancer. In lung cancer is more common in small cell cancer than in non-small cell cancer. In small cell cancer is seen in 80% of cases.

In small cell cancer is a close connection between smoking and frequency, type and appearance of the mutation, with a prevalence of G-T(1) transversions.

Downstream the p53 pathway more target genes are disordered, the most frequent being the Bcl 2-Bax pathway and ratio between them (20,21).

Bcl 2 is negatively transcriptionally regulated, unlike Bax, which is positively regulated by the p53 overregulation of Bcl 2 (which has antiapoptotic action) and Bax subregulation (which has proapoptotic action) is more common in small cell cancer than in non-small cell cancer and confirms us the resistance to apoptosis in small cell cancer.

Another transcriptional target-gene of the p53 is FAS. This is a member of the transcriptional protein superfamily, Tumor necrosis factor (TNF).

In contact with the FASL ligand, FAS induces apoptosis by means of intracellular signaling pathways which include Caspase-8 and FADD (FAS-associated death domain).

The FAS pathway alteration leads to resistance to apoptosis in many human solid tumors including lung cancer (21,22).

The loss of FAS expression on the cell surface in neural endocrine tumors correlates with the cellular resistance  compared to FAS-mediated apoptosis (23)

In histochemical studies are seen in the case of small cell cancer, a severe reduction or absence of FAS expression, as compared with normal cells in 94% of cases.

In contrast, FASL is highly expressed in 90% of small cell cancer cases, and in adenocarcinoma and squamous form is underexpressed - as compared to normal - in 70% of cases.

The presence of FASL overexpression and FAS underregulation in small cell cancer sugsests that tumor cells escape from the suicidal action. This makes that the cells of the small cell cancer induce the paracrine killing of cytotoxic T cells that express FAS. This is also confirmed by the small number of lymphocytic infiltrates and in particular cytotoxic T cells.

Caspase-8 is methylated in the cell lines of lung cancer and can not contribute anylonger  to the induction of apoptosis (24).

  RETINOBLASTOMA  ALTERATIONS IN  LUNG CANCER

Retinoblastoma (Rb) is a tumor-supressing gene, which encodes a nuclear protein that acts as a control point in the cell cycle in phase G according to the state of phosphorylation, which is in part regulated by p53. The stopping in G phase is accomplished by the hypophosphorylated form of Rb, which is fixed on E2F1 and represses its transcriptional activity.

Rb is thus the major target-pathway of cell cycle regulation (25).

The Rb(pRb) proteine along with p16INK4 and CyclinD1 are the major components of the Rb pathway, which controls the transition from phase G1 to phase S within the cell cycle.

p21 is an inhibitor of the cyclin-dependent kinases (CDK). By this is prevented the Rb phosphorylation, allowing the stop in G1 phase.

Conversely, cyclin D1 together with CDK4 and CDK6 perfect the phosphorylation of Rb, with the release of the E2F1 transcription factor, which is followed by the transition from the G1 phase to S phase, which leads to an increased proliferation.

p16INK4 is a tumor-supressing gene. It is an inhibitor that prevents the association of CDK4 and CDK6 cyclin-dependent kinases with D-type cyclines.

The loss of p16INK4 expression is an early event and is correlated with reduced survival (2-26).

In small cell cancer we have a reverse relation between Rb and p16INK4.

In small cell cancer we have a direct relation between Rb and cyclinD1.

All these shows us that Rb is the major pathway target of the cell cycle regulation (1).

Small cell cancer and neuroendocrine cell cancer have do not color in nuclear (immunohistochemical) terms for Rb protein.

Small cell cancer and endocrine cell cancer have abnormalities of the Rb şi and p16INK4 pathways.

Carcinoidds are couloured positively nuclear for Rb. Typical carcinoids have normal pathways, Rb and p16INK4. Atypical carcinoids have partial abnormalities of the p16INK4 or Rb (2,27,28).

The intact Rb proteine is seen only in 10% in small cell cancers.

Overexpression of cyclinD1 is noticed only in 1,3% of small cell cancers.

The p16INK4 loss is only seen in 7% of small cell cancers.

Overexpression of cyclinE with the concomitant loss of Rb is observed in 30% -40% of small cell cancer (1,29).

The Rb gene activation mechanism in small cell cancer is not well known, mutations being more frequent on cell lines than in tumors. There have been detected only 25% mutations - in small cell lung cancer tumors-located in exons 13-18 or exons 20-24 of the Rb gene (1,29).

ALTERATION OF THE p14ARF AND MDM2(REGULATORS OF THE p53 PATHWAY)

p14ARF is a product al of the CDKN2(INK4a) locus. Along with p16INK4a is involved in cell cycle regulation.

p14ARF inhibits MDM2, promotes p53, which in turn acts on p21, which fixing to Cyclin-CDK complexes, delays the cell cycle progression and induces apoptosis in a dependent way (30) or/and independently of p53 and MDM2 (1).

The loss of p14ARF through homozygous mutations of the CDKN2A (p16INK4a) gene leads to an increase in the level of MDM2, which induces the loss of p53 function and loss of cellular control.

MDM2 (murine double minute2) is an oncogene. MDM2 acts on p53 namely by antagonizing its transcriptional activity and promotes its degradation (31,32,33). This inhibitory effect on p53 is counter-attacked by p14ARF(34), which, fixing on MDM2 inhibits the  p53 degradation (35). p14ARF prevents the regulation through negative feed-back by MDM2  on p53.

MDM2 was found expressed in 30% of cases of small cell cancer and other neuroendocrine cancers accompanied by the exclusion of p14ARF gene.

Between p14ARF and MDM2 there is an inverse relation. The ratio MDM2/p14ARF higher than 1 shows a tumor phenotype with a neuroendocrine component.

There is also an interaction between MDM2 and Rb, namely: MDM2 inhibits the Rb regulation function through a mechanism still unknown. The overexpression of MDM2 and (or) loss of p14ARF in small cell cancer alters the p53 and Rb functions allowing the inactivation of p53 and Rb pathways. Thus is favored the evasion of stopping in phases G2 and G1 of the cell cycle (1) and we therefore have cell proliferation.

THE E2F1 OVEREXPRESSION.

E2F1 is a transcription factor and is a key component of the cell cycle, which acts through the transactivation of genes necessary for entring into S phase

E2F1 is overexpressed in small cell cancer in 92% of cases, and is undetectable in 90% of the cases of non-small cell cancer (36). In small cell cancer, in contrast with non-small cell cancer, overregulation of E2F1 is related to nuclear accumulation and an overexpression of multiple target genes. E2F1 is overexpressed in small cell cancer and large cell neuroendocrine cancer and is related to a high Ki-67 index and a ratio Bcl2/Bax>1. It was not observed an enhancement of E2F1 (36). The Bcl2/Bax>1 ratio is correlated with a low apoptotic index in neuroendocrine tumors. The ratio is higher in small cell cancer compared to non-small cell cancer. This ratio correlates with overregulation of E2F1 in small cell cancer. In non-small cell cancer we have a Bcl2/Bax<1 ration and a low expression of E2F1.

E2F1 is involved in human carcinogenesis and is released by Rb phosphorylation.

The E2F1 overregulation is likely to be responsible for cyclin E overregulation in small cell cancer, where there is a lack of Rb, as cyclin E is one of the transcription target of E2F1. Disturbance of E2F1 in neuroendocrine lung tumors suggests that this disorder is involved in carcinogenesis (36).

During tumorigenesis E2F1is involved both as oncogene (Johnson in 1993, quote 36) or tumorsupressing gene (Pierce in1999, quote 36), this depending on the context and the ability to induce cell proliferation and (or) or apoptosis dependent or not on p53.

In small cell cancer E2F1 acts as oncogene, is overregulated, the small cell cancer is the most proliferative and aggressive form of lung cancer. As oncogene leads to uncontrolled proliferation probably by counteracting the action of tyrosine-kinase inhibitors.

As a future therapeutic option, we have to neutralize the E2F1production in small cell cancer to inhibit proliferation and in non-small cell cancer-inverse must induce the E2F1 expression to restore apoptosis (36).

Deletions to 3P chromosome

Most small cell cancers have allelic losses in 3p segment. This is an area rich in genes. Genes showing LOH (loss of heterozygozity) are usually tumor suppressing genes.

Among the genes located in the 3p region,  involved in small cell cancer, we distinguish:

-            The FHIT(3.14.2) gene is involved in the regulation of apoptosis and the cell control (37). The FHIT gene is subregulated (loss of the FHIT protein expression) in over 80% of small cell cancers (38). This forwards the cell proliferation.

-            The SEMA3F (class 3 semaphorin) gene is very frequently altered in lung cancer and in particular in small cell cancer. It is located in the 3p.21.3 and loses function in the 3p deletions area.

-            The RASSF 1(Ras-associated domain family 1A) gene is located in the 3p.21.3 and is a candidate tumor suppressing gene. It also intervenes in cell cycle progression through interaction with the Rb pathway. In lung cancer undergoes allelic losses. The gene shows 2 products (1A and 1C), of which RASSF1A undergoes an epigenetic inactivation in lung cancer by hypermethylation of the region concerned, and in particular in small cell cancer reaching 90% of cases (39). Normally it has the ability to suppress also the growth on experimental data - it has antitumorigenic action (39,40).

-            The SEMA 3B gene is also located in 3p.21 area and is potentially a tumor-suppressor gene. It has been found its methylation in non-small cell cancer, but this has not been observed in small cell cancer.

-            Another tumor-suppressor gene located in the 3p.24.26 area is RAR-beta. The retinoic acid has a role in lung development and cell differentiation by its interaction with nuclear retinoid receptors encoded by the RAR-beta or RAR-XR gene. It was observed in 72% of cases of small cell cancer an epigenetic inactivation by methylation of RAR-XR(40).

TELOMERASE EXPRESSION

Human telomerase reverse transcriptase is a ribonucleoprotein that synthesizes telomeric sequences, which decreases with each cell division. In cancer cell this activity leads to unlimited (immortal) proliferation.

Almost all small cell cancers have a high telomerase activity (41,42,43). Telomere shortening acts as a mitotic clock.

Cells telomerase activity is expressed by hTERT, which is a reverse transcriptase catalytic subunit. There is a concordance between the hTERT protein expression and telomerase activity detection.

Telomerase activity can be used as a diagnostic marker. In small cell lung cancer, telomerase activity is low in its squamous form and very low adenocarcinoma . Instead is very high in small cell cancers, large cell cancer, basaloid form, and neuroendocrine cell lung cancer.

In immuno – histochemical terms is observed a diffuse and intense nuclear small cell cancer and in basaloid form of the large cell cancer, while in adenocarcinoma and squamous form of the non-small cell cancer is seen a nuclear or nucleolar staining (44 ) . The telomerase expression is lower in stage I and increases along with the extension of disease. Nucleolar staining is also correlated with a shorter survival.

Thus, the telomerase expression is distinct among the histo-pathological subsets of lung cancer and influences prognosis.

FACTORII  ANGIOGENICI

VEGF (vascular endothelial growth factor) is a signaling protein involved in vasculogenesis (formation of the circulatory system) and angiogenesis (vascular growth from pre-existing vessels). Its activity is restricted only to endothelial cells.

All members of the VEGF family stimulate the cellular response by fixing tyrosine-kinase receptors from the cell surface. There are three tyrosine-kinase receptors on the cell surface: VEGFR-1(Flt-1), VEGFR-2(Flk-1(KDR)  and VEGFR-3(Flt-4)(45,46). VEGFR-2 seem to mediate almost all cellular responses known.VEGFR-1 and VEGFR-2 have a role in the normal development of the lung and maturation of the alveolar epithelial cells (47).

VEGF is secreted by the tumor cells. Also the receptors VEGFR-1 and VEGFR-2 are expressed in tumor cells (1).

 There have also been discovered further 2 receptors of VEGFR-2: neuropilin-1 and neuropilin-2 (NP1 and NP2) which have been found in neuronal cells and endothelial cells and are expressed in various tumor cells. VEGF may be involved either directly through an autocrine manner, or indirectly by NP1 and NP2 receptors (48).

Several tumor cells, including lung cancers, express VEGFR-1,VEGFR-2, as well as NP 1 and NP 2(48).

Another neuropilins ligand is SEMA 3F, which is an antagonist of VEGF.

SEMA 3F was isolated from the 3p region, the region where there are losses of heterozygosity in lung tumors. SEMA3F has a tumor suppressor activity, which is mediated by the reduction of the integrin activity, of the adhesion to extracellular matrix components and lowering of MAPK signaling pathway phosphorylation (49). SEMA 3F also has an apoptotic and antimigratory role. It plays an important role in cell adhesion. Loss of SEMA 3F in small cell cancer is significant and contributes to the pathogenesis of small cell cancer (1).

SEMA3F links to NP1 şi NP2 and splits with VEGF this connection, becoming an antagonist of the VEGF. There is thus a competition between VEGF and SEMA3F for binding to NP1 and NP2receptors.

The SEMA3F loss and VEGF165 gain leads to tumor growth. SEMA3F loss is significant in small cancer cells and contributes to its pathogenesis. This loss is early in small cell cancer and is related to an increased tumor aggressiveness (49).

The early alteration of the VEGF-SEMA3F-NP pathway is seen during the lung cancer progression (48).

THE CADHERIN-CATENIN COMPLEX PROTEINS

Small cell lung cancer is characterized by the invasive growth of tumor cells , with a tendency to metastasize early, making it contra-indicated for surgery.

E- cadherin is a member of the CADHERIN family that mediates calcium-dependent cell adhesion. It is a transmembrane glycoprotein. It plays an important role in carcinogenesis and tumor invasion.

Beta catenin is a cytoplasmic protein which interacts directly with E- cadherin. It links this molecule to actin-cyto-skeleton, via alpha-catenin. The cell adhesion function depends on the integrity of the entire network: E-cadherin, beta-catenin , actin (1). The regulation lowering of E-cadherin leads to decrease differentiation and increases the tumor aggressiveness and metastasis frequency.

In neuroendocrine tumors has been ovserved a very common alteration of E- cadherin expression and Beta Catenin in 90 % of cases. In small cell cancer Beta- Catenin does not stain. This is in contrast to the carcinogenesis of colorectal cancer where there is an activation of Beta-Catenin (50,51,52).

In small cell cancer Beta-Catenin is located in the area of 3p deletions, namely in  3p21.3 area, having rather a role of tumor supressor gene, than oncogene(1).

THE GROWTH FACTOR OF TYROSINE-KINASES AND ITS RECEPTORS

Tyrosine-kinase receptors are key-molecules in normal cellular differentiation. They are involved in the etiology of malignant tumors. They are usually disordered or mutated in human cancers and are attractive target molecules within the selective inhibitor therapy (53).

- The KIT tyrosine/kinase receptor along with the PDGF (platelet derived growth factor) receptor  facilitate (lead) an intra cytoplasmic signal cascade which in turn leads to cell growth (54,55,56,57).

The activation of c-KIT tyrosine-kinase through stem cell factor is one of the mechanisms proposed to explain the pathogenesis of small cell cancer (58). 70% of small cell cancers express the KIT tyrosine kinase receptor and its ligand, STEM CELL FACTOR(SCF)(59). There are not known many data on the PDGF receptor in small cell cancer cells (1).

The action of KIT/SCF may be inhibited by means of an inhibitor of tyrosine-kinase named STI571 which inhibits the KIT activation mediated by SCF(59). STI571 also inhibits the MAPK and AKT activation mediat by SCF, but has no effect on Insulin-Like Growth Factor. It is an inhibitor of growth. It blocks or slows relapse after chemotherapy. It can be administered along with Carboplatin and Etoposide (59,60).

    - MET is the product of c-MET proto-oncogene and is a receptor-kinase. Together with its ligand, hepatocyte growth factor (HGF) are involved in epithelial-mesenchymal transition in proliferation, cell life increase, in angiogenesis, activates cell motility, raises the invasion and is over expressed in many solid tumors (58). The aberrant activation of MET causes a cascade of cytoplasmic signals that lead to further activation of multiple signal transduction and transcription (58,61,62,63). c-MET appears to be activated in a paracrine way, while HGF is produced by the stromal cells.

c-MET over expression and mutations may lead to carcinogenesis in multiple tumors. In lung cancer we find a high level of c-MET-mRNA. In small cell cancer is expressed and phosphorylated. The MET / HGF pathway contributes to the invasive phenotype developments and has an important role in oncogenesis of small cell lung cancer (58). The pathway activity of hepatocyte growth factor / c-MET may be inhibited by means of compound Geldanamycin that reduces the phosphorylation of c-MET proteins. This leads to the inhibition of growth, cell viability in small cell cancer, leading to apoptosis (58).

CONCLUSION,

-              small cell lung cancer is a malignant tumor with small cell and a very high nuclear-cytoplasmic ratio.

-              Precursors are not known. It simultaneously belongs to the neuro-endocrine tumors which are highlighted through histo-chemical methods and data from the electron microscopy.

-              Although responds very well to treatment, the survival time is short. In order to be termed small cell cancer it has histologically show at least 10% injuries with the appearance of small cell cancer.

-              Small cell cancer is a proliferation that arises from the bronchial basal cells. It has a high proliferation rate. It is an aneuploid neoplasm.

-              TTF-1 is present at a high level diagnostic value for differentiating it from the basaloid carcinoma.

-              From three neuroendocrine markers, the most sensitive for diagnosis is CD56(NCAM)

Molecular genetic changes in the pathogenesis of small cell cancer.

-              The SHH pathway induces the neuroendocrine differentiation and is activated and involved in the malignant phenotype of small cell cancer.

-              hASH transcription factor, involved in neuroendocrine differentiation, is present at high levels in small cell cancer.

-              The p53 gene mutations are present in 80% of small cell cancers cases.

-              The gene Bcl 2 – an antiapoptotic gene - is overregulated and the pro-apoptotic Bax gene is subregulated in small cell cancer.

-              The FAS transcriptional gene that induces apoptosis is reduced in small cell cancer. Instead the ligand (FASL) is highly expressed. This leads to the tumor cells escape from apoptosis and contributes to proliferation.

-              RETINOBLASTOMA is the major pathway of regulating the cell cycle. pRb together with p16INK4 şi cyclin D1 are the major components of the Rb pathway that controls the transition from the G1 phase to S phase of the cell cycle. In small cell cancer we have an inverse relation between Rb and p16INK4a and direct relation between Rb and cyclin D1. Small cell cancer and neuroendocrine cell cancer have abnormalities of the Rb and p16INK4 pathways.

-              Regulators of the p53 pathway,  p14ARF is involved in regulating the cell cycle.

-              MDM2 is an oncogene which acts negatively on the p53gene. This inhibitory effect is counteracted by p14ARF, which are attached to MDM2 and inhibits the p53 degradation

-              The MDM2/p14ARF>1 ration shows a tumor phenotype with neuroendocrine component. MDM2 overexpression and (or) p14ARF loss of small cell cancer lead to the p53 and Rb pathways inactivation, which leads to the increase of proliferation

-              E2F1, transcription factor, is a key component of the cell cycle. It is overexpressed in 92% of small cell cancer cases. It is associated with a high Ki-67 and a Bcl 2/Bax>1 ratio. E2F1 is responsible for overregulating the cyclin E in small cell cancer where Rb lacks. The E2F1 disorder in small cell cancer suggests its involvement in carcinogenesis.

-              The genes located in the 3p area and are involved in small cell cancer, are the following:

-          FHIT gene is underregulated in 80% of small cell cancer cases, facilitating the cell proliferation.

-          SEMA 3F gene is frequently altered in small cell cancer.

-          RASSF1 gene is inactivated in 90% of small cell cancer cases.

-          RAR-Xr gene is inactivated through metilation in is inactivated.

-              The telomerase activity is very high in small cell cancer. Telomerase expression increases along with the disease extension. Immuno-histochemically there is a diffuse nuclear staining in small cell cancer

-              VEGF is a signaling protein involved in vasculogenesis and angiogenesis and stimulates the cell response by fixing on the tyrosine-kinase receptors of cell surface. VEGF, VEGR-1, VEGFR-2 are secreted by tumor cells.

-              There are also 2 receptors, NP1 and NP2, which are expressed by the tumor cells. Cells in lung cancer express VEGF and tyrosine-kinase receptors.

-              There is also a ligand of the SEMA3F neuropilins, which is an antagonist of the VEGF. It has and apoptotic and anti-migratory role. There is a competition between VEGF and SEMA3F for connecting to the NP-1 and NP-2 neuropilins.

-              The loss of SEMA3F in small cell cancer is significant and contributes to the pathogenesis of small cell cancer and is related to an increased aggression.

-              The E-cadherin and beta-catenin complex mediates cell adhesion. In tumors with neuroendocrine component is altered the expression complex in 90% of cases.

-              The KIT tyrosine-kinase receptor together with the PDGF receptor are inactivated and induce a cascade of intra-cytoplasmic signals leading to cell growth. 70% of small cell cancers express the KIT tyrosine-kinase receptor and its ligand SCF (stem cell factor). The KIT activation through SCF is one of the mechanisms of pathogenesis of small cell lung cancer

-              MET(the product of c-MET proto-oncogene) is a tyrosine-kinase receptor and has HGF as ligand (Hepathocyt growth factor). The MET/HGF pathway contributes to the development of invasive phenotype and plays a role in the oncogenesis of small cell cancer by its aberrant activation (overexpression or aberrations).

SYNTHESIS

FACTORS FACILITATING

PROLIFERATION IN SMALL CELL LUNG CANCER

Micro-cell carcinomas have a high rate of proliferation. This is highlighted by determining the Ki-67 index.

1)    The SHH signaling pathway is activated in small cell lung cancer and is involved in developping the malignant phenotype and in neuroendocrine differentiation.

2)    The hASH transcription factor is present at high levels, promotes the epithelial airway proliferation and enhances the tumorigenesis, when losing the functions p53 and pRb.

3)    The p53 gene mutations is very common in small cell lung cancer. This leads - downstream - to the disorder of several target genes including:

-  overregulation of Bcl 2, underregulation of Bax and increase of Bcl/Bax ratio,

- alteration of  FAS pathway, leads to resistence towards the apoptosis mediated  by it (FAS),

- FAS underregulation and FASL (ligand) overexpression favors the proliferation.

4)    Caspase-8 methylation impedes apoptosis, leading to proliferation

5)    Rb phosphorylation through the summarized action of cyclinD1 together with CDK4 and CDK6 leads to the release of the transcription factor E2F1, which is followed by the transition from the G1 phase to the S phase of the cell cycle, leading to proliferation. To these can be added the loss of p16INK4A expression, which in turn may only hamper the association of cyclin-dependent-kinases CD4 and CD6 with cyclines of type D and overexpression of cyclinE.

6)    The loss of p14ARF through homozygous mutations leads to an increase in MDM2, which further induces the p53 loss of function and therefore loss of cell cycle control. Thus is enhanced the proliferation.

7)   The E2F1 transcription factor is overexpressed in small cell lung cancer in 92% of cases and practically not observed in non-small cell lung cancer. It is released by phosphorylation of Rb. Simultaneously with the absence of Rb leads to cyclinE over regulation.

E2F1 acts as overregulated oncogene in small cell lung cancer, leading to an aggressive proliferation, probably by counteracting the tyrosine-kinase inhibitors.

8) Loss of the FHIT protein expression (underregulation), SEMA3F gene alteration, alteration by hypermethylation of the tumor suppressor gene RASSF1A, RAR-XR gene inactivation lead to cell proliferation.

9) Telomerase activity is very high in small cell lung cancer, activity that increases along with the disease stage, compared to non-small cell lung cancer. It is a diagnostic marker.

10) The angiogenic factors involved in small cell lung cancer are:

The VEGF signaling protein involved in vasculogenesis and angiogenesis. Family members stimulate the cell response by determining the tyrosine-kinase receptors from the cell surface VEGFR1 and VEGFR2. VEGFR2 also shows 2 receptors Neuropilin1 (N) and neuropilin 2 (NP2). All are expressed in tumor cells. In addition there is a SEMA3F which is a ligand of neuropilins and VEGF antagonist. It has a tumor-suppressor action. It is an antagonist of VEGF, binding to the NP1 and NP2 neoropilins. In small cell lung cancer, the SEMA3F loss and VEGF165gain lead to tumor growth and aggressiveness.

11)  the altered expression of E-Cadherin Beta-Catenin is frequent in 90% of cases of small cell lung cancer. The regulation lowering of E-cadherin leads to decrease differentiation, increases tumor aggressiveness and frequency of metastasis. The cell adhesion phenotype depends on the network integrity.

12)  The tyrosine-kinase receptors are key molecules in cell differentiation.

In small cell lung cancer 70% of the cases express the KIT tyrosine-kinase receptor and its ligand Stem Cell Factor (SCF). Kit activation by SCF is one of the mechanisms of small cell lung cancer pathogenesis.

MET, the oncogene product of c/MET is a tyrosine /kinase receptor, which together with its ligand, HGF, are involved in the epithelial-mesenchymal transition, in proliferation. It is overexpressed and phosphorylated in small cell lung cancer. The MET /HGF pathway contributes to the invasive phenotype development and has role in small cell lung cancer oncogenesis.

GLOSSARY

MYCL1      is a transcription factor involved in lung cancer with localization in the area 1p34.3.

GL1-           gliom associated oncogene homolog-1(zinc finger protein) Is a transcription factor activated by the Sonic Hedgehog signaling pathway, which acts on the stem-cell compartment and induces the neuroendocrine differentiation. The Sonic Hedgehog pathway is activated in small cell cancer.

SSH            (SONIC HEDGEHOG HOMOLOG-1 is a protein from the Hedgehog signaling pathway. In adults controls the cell division of stem cells cycle and is involved in the development of some cancers. There is a Hedgehog inhibitor named Robotnikin.

hASH-1       (human achaete-scute homolog-1) is a transcription factor crucial to the development of sympathetic nervous system. It is present in the cells of small cell lung cancer, lacks in non-endocrine cancers.

CD99          is a human surface glycoprotein encoded by the CD99 gene. It is expressed in all leukocytes, especially in tymocites. Increases T cells adhesion and apoptosis and participates in migration and activation.

Graninele     (chromogranin and secretogranin ) are a family of proteins, which are found in a variety of endocrine and neuroendocrine cells.

Synaptophysin is a membrane glycoprotein. Is present in neoplasms with neuroendocrine component.

CD56          (neural cell adhesion molecule NCAM) is a glycoprotein expressed on the neurons surface, Naturall Killer cells, T cells activated in the endocrine tissue. It is present in small cell lung cancer, along with other neuro-endocrine neoplasms.

Tumor Antigenes -  p53, CAGE1, N Y-Eso-1, GBU4-5,  ANNEXIN, SOX2, HU-D.

CASPASE 8. Is a member of the  Cysteine-Aspartic Acid ​​Protease (caspase) family. Plays a major role in the execution phase of cell apoptosis induced by FAS and other apoptotic stimuli.

FAS receptor is an important cell surface receptor protein, which is part of the TNF family (tumor necrosis factor) and is also known under name CD95. Induces apoptosis by attaching to the FAS LIGAND.

p14ARF      is a product of the CDKN2A locus.

p16INK4A and p14ARF are involved in cell cycle regulation. p14ARF inhibits MDM2, promoting p53, which activates p21, which in turn inactivates CDK (cyclin-dependent-kinase).

Cyclin          –dependent – kinases usually promotes transcription genes, leading (pushing) cell cycle to S phase. The loss of p14ARF through homozygous mutation and of the p16INK4A gene leads to the increased expression of MDM2, which further leads to the loss of p53 function, leading to the loss of cycle cell control.

FHIT           Fragile hystidine triad protein is a tumor supressor gene. It acts simultaneously with the VHL gene, another tumor-suppressor gene that protects the appearance of chemical etiology lung cancer.

SEMA3F     is a protein secreted by the SEMAPHORIN 3 family. They are signaling molecules.

RASSF        is a gene with function of tumor-suppressor gene. Its altered expression of the mutations is related to its pathogenesis of some cancers.

RAR-beta is part of a superfamily of nuclear regulators of transcription. It attaches to the retinoic acid and interferes with cell growth and in differentiation.

FLT-1 D3    human(VEGFR-1 D3 recombinant) is a protein encoded by the FLT-1gene. It is part of the SRC gene family. Has a tyrosine-protein kinase activity. Controls the cell proliferation and differentiation.

KDR           (kinase insert domain receptor) is a type III tyrosine-kinase receptor. Is known as VEGFR-2 kinase, as CD309or as FLK-1 (fetal liver kinase)

CD           (cluster differentiation). It is a protocol for the classification of cell surface molecules present on leukocytes, being targets for immuno-phenotyping of cells. Act as receptors or ligands (the molecule that acts on receptors). Intervene in signaling and adhesion.

FLT4.          Is a protein encoded by the FLT4 gene. This gene is a tyrosine-kinase receptor for VEGF- C şi VEGF- D, which are involved in lymphangiogenesis.

NRP (neuropilin)-1. Is a protein encoded by the NRP-1 gene. Is a receptor protein, which binds to  VEGF and SEMAPHORIN.

NRP(neuropilin)-2. Is a receptor protein encoded by the NRP-2 gene. It binds to the SEMA 3F protein and interacts with VEGF. It has a role to developments in cardio-vascular tumorigenesis and in axon guidance.

MAPK is a serine-threonine protein kinase involved in the gene expression, mitosis, differentiation, proliferation, cell survival, apoptosis.

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